Nanopore adaptive sampling to identify the NLR gene family in melon (Cucumis melo L.)

Abstract

Background: Nanopore adaptive sampling (NAS) offers a promising approach for assessing genetic diversity in targeted genomic regions. Here we designed and validated an experiment to enrich a set of resistance genes in several melon cultivars as a proof of concept.

Results: Using the same reference to guide read acceptance or rejection with NAS, we successfully and accurately reconstructed the 15 regions in two newly assembled ssp. melo genomes and in a third ssp. agrestis cultivar. We obtained fourfold enrichment regardless of the tested samples, but with some variations according to the enriched regions. The accuracy of our assembly was further confirmed by PCR in the agrestis cultivar. We discussed parameters that could influence the enrichment and accuracy of NAS generated assemblies.

Conclusions: Overall, we demonstrated that NAS is a simple and efficient approach for exploring complex genomic regions, such as clusters of Nucleotide-binding site leucine-rich repeat (NLR) resistance genes. These regions are characterized by containing a high number of copy number variations, presence-absence polymorphisms and repetitive elements. These features make accurate assembly challenging but are crucial to study due to their central role in plant immunity and disease resistance. This approach facilitates resistance gene characterization in a large number of individuals, as required when breeding new cultivars suitable for the agroecological transition.

Keywords: Genome assembly; Melon; NLR; Nanopore adaptive sampling; Resistance genes; Targeted sequencing.

Fig. 1

Schematic representation of the definition of ROIs, target regions and target regions without repetitive elements (REs). Target regions without REs were provided to the MinKNOW software. Regions were defined in the reference genome for NAS, Anso77

Fig. 2

shows a workflow diagram summarizing the different steps involved in the data processing and target region assembly process. All statistical analyses were performed using R v. 4.1.1 [51]

Fig. 3

Read length distribution of the different generated datasets. (A) Length distribution of “PASS”-tagged NAS reads. The number of reads was log-transformed. (B) Length distribution of WGS reads after filtering by “end reason”, quality and length. (C) Length distribution of NAS reads after filtering by “end reason”, quality and length

Fig. 4

NAS sequencing depth on three representative target regions of different sizes. (A) NAS sequencing depth on the three target regions compared to the rest of the chromosome. Target regions (ROI + 20 kb buffer) are represented between black dotted bars, while ROIs are collapsed and represented between black solid bars. (B) NAS sequencing depth on the ROI of region 01 of chromosome 1 (≈ 173 kb). (C) NAS sequencing depth on the ROI of region 08 of chromosome 5 (≈ 998 kb) (D) NAS sequencing depth on the ROI of region 12 of chromosome 8 (≈ 1 kb). Region 08 contains the well-studied Vat cluster. For B, C and D, vertical colored bars represent the enriched regions, while vertical white bars represent masked repetitive elements

Fig. 5

Enrichment by yield (A, C) and enrichment by selection (B, D) of the 15 target regions from Anso77 (A, B) and Doublon (C, D). Vertical red-dotted bars denote the flowcell washing flush time

Fig. 6

Dot plots representing the Vat region for Anso77 (A) and Doublon (B). Reference Vat region is represented on the x-axis, while the NAS-reconstructed Vat region is represented on the y-axis

Fig. 7

Sequence depth by time in the NAS experience of the 15 target regions from Chang-Bougi

Fig. 8

Dot plots representing the NAS filtered assembly of Chang-Bougi (y-axis) against two sequences. (A) NAS filtered assembly of Chang-Bougi (y-axis) against the 15 target regions from Anso77 (x-axis). (B) NAS filtered assembly of Chang-Bougi (y-axis) against the 18 NLR clusters identified in the Chang-Bougi assembly published by Shin et al. [33]

Fig. 9

Manual annotation and validation of the Vat region of Chang-Bougi. (A) Genes identified after manual annotation within the Vat regions of Chang-Bougi. The sequence above was obtained from the NAS assembly, while the sequence below was recovered from the publicly available genome assembly [33]. (B) Agarose gel electrophoresis of PCR products obtained using primers Z649FR and Z1431FR. Lanes M1 and M2 are the two 1 kb DNA ladders (Promega, Madison, WI, USA). PI161375 was used as a control having a Vat1 with four R65aa motifs and a Vat2 with three R65aa motifs. Bands pointed with arrows represent an amplicon of four R65aa motifs (1), an amplicon of three R65aa motifs (2), and a specific amplicon of four R65aa motifs (3)

Fig. 10

Comparison of the use of standard and ultra-long reads in the enrichment of the Vat cluster. The diagram illustrates the difference in coverage and extent of the area outside the region to be enriched for standard size fragments (10-30 kb) and for ultra-high molecular weight fragments (100-300 kb) on the Vat (melon) cluster. For the same yield (illustrated here by an arbitrary overall depth of 5X), standard fragments make it possible to achieve a depth more concentrated on the area to be enriched and to sequence less outside this area. For convenience, only reads oriented from 5’ to 3’ are represented